IE67888B1 - Novel derivatives of endogenous mediators their salts method of preparation applications and compositions in which they are present - Google Patents

Abstract

Derivatives of biologically active molecules containing a primary amine group and a hydroxylated nucleus, and their addition salts with mineral or organic acids, having general formula I: R'R"N-A-B-O-CH2-CO-NH-R(I) A=linear or branched C1-C5 alkylene; B=C4-C10 aromatic nucleus with optional heteroatom, or=group -B1-X-B2-, wherein X=O or C1-C4 alkylene; -NH-R=amine residue or alcohol residue; and R', R"=C1-C5 alkyl, H or hydrophobic radical, and their addition salts with mineral or organic acids, method of preparation, applications, especially to visualization, purification, the preparation of antibodies and the assay of the total hormone, and as drugs, compositions in which they are present and analytical kits.

Description

Novel derivatives of endogenous mediators. their salts. method of preparation, applications and compositions in which they are present The present invention relates to novel derivatives of endogenous mediators, to their salts, to their method of preparation, to their applications, especially to the analysis of endogenous mediators, the analysis or purification of the receptors for said mediators, the visualization of their receptor sites, the preparation of antibodies and the use of said antibodies for the analysis of endogenous mediators, to their application as drugs and to compositions in which they are present.

Messages are transmitted between cells by way of chemical vectors (hormones or neuromediators) whose specificity is assured by the target protein: the receptor. The molecules carrying the information can be peptides or low molecular weight molecules. A'num20 ber of these simultaneously possess one or more hydroxyl groups and one or more amine groups, for example indolamines, catecholamines (neuromediators) and thyroxine (hormone).

These molecules play a role of prime importance in physiology: indolamines and catecholamines in the transmission and integration of information in the central and peripheral nervous systems and thyroxine in the regulation of the basal metabolism. It is essential to assay them for the purpose of improving diagnoses or assisting functional researches. In the case of neuromediators, the receptor binding sites assure the specificity of the response of the effector cell: localized chemical modification of the endogenous ligands makes it possible to distinguish between the various types and subtypes of membrane receptor. Thus modified ligands could, be drugs acting specifirally an the physiological functions controlled by these receptors.

V s Certain hormones are present in the serum in a fora bound to carrier proteins. These proteins ensure that these horaoiies are transported from the point of synthesis to the target cells. The thyroid hormones are present in an unbound fora at levels of less than 1% of the total amount of hormones (free + bound). Localized chemical modification of the thyroid hormones enables their assay to be improved.

The Applicants have discovered that these 10 objectives can be achieved through O-carboxymethylation on the hydroxyphenyl group of endogenous molecules also carrying a primary aipine.

Carboxymethylation (Gurd, Methods in Enzyrnology, vol. XI, 1967, p. 532-541) has often been used to block the amine groups of amino acids. The carboxymethylation of hydroxyphenyl groups is described as a .side .,3reaction (Koraan and Clarke, J. Biol. Chem., 221, 1956, p. 113-131). The carboxymethylation of hydroxyphenyl groups (Spector, 1982) has been used for coupling morphine (which does not possess an amine group) with a protein to give an antibodyi It is for this reason that the. present patent application relates to novel derivatives of biologically active molecules containing a primary amine group and a hydroxylated nucleus, characterized in that they have the’general., .formula I : [R'R-N-A-B-O-CH2-CO]n-Rj (I) in which 3Q n represents an integer from 1 to 10; A is a linear or branched alkylene chain containing from 1 to 5 carbon atoms; B is an aromatic nucleus containing from 6 to 10 carbon atoms, if appropriate substituted, and optionally containing j a heteroatom, .......

Rj is an amine residue or an alcohol residue chosen from among optionally substituted phenols and Cj-Cjg aliphatic alcohols, R' and R are an alkyl radical containing from 1 to 5 carbon atoms or a hydrogen atom, as well as their addition salts with mineral or organic acids.

The additional salts with mineral or organic acids can be, for example, salts formed with hydrochloric, hydrobromic, nitric, sulphuric, phosphoric, acetic, formic, propionic, benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, glyoxylic and aspartic acids, alkanesulphonic acids such as methaneor ethane-sulphonic acid, arylsulphonic acids such as benzene- or paratoluene-sulphonic acid, or arylcarboxylic acids. It is -preferable.to . use the salts of chaotropic anions, such as citrate or succinate ions, which are capable of facilitating dissolution in aqueous media.

The alkylene chain represented by A can be a propylene, isopropylene, methylene or, preferably, ethylene chain; it can be substituted by a sulphydryl group.

The aromatic nucleus can contain a single ring, such as a phenyl nucleus, or two rings, such as an indole nucleus for example. One or more of the carbon atoms of B can be substituted by a radical selected from halogen atoms such as chlorine, alkoxy radicals such as ethoxy or, preferably, methoxy, alkyl radicals such as propyl, ethyl or, preferably, methyl, alkylthio radicals such as methyl thio, and polyhalogenoalkyl radicals such as trifluoromethyl.

The hydroxyl substituted by the radical -CHj-CO-NH-R according to the present invention can be located in any position, but is located particularly on a phenyl ring of the nucleus and preferably in the positions which this hydroxyl radical normally occupies in natural mediators.

If R' and R" are an alkyl radical, they are preferably a methyl or ethyl radical. Rz and R are advantageously hydrogen. Aminoacyl radical is understood as meaning amino acid residues, peptides or proteins. Hydrophobic radical is understood as meaning that said radical possesses saturated chains and does not contain groups such as free amino or hydroxyl groups.

Rz and R are advantageously a hydrogen atom or a methyl or ethyl radical, it being possible for the radical NRZR to be quaternized, if appropriate, and examples which may be mentioned are trimethylammonium or diethylmethylamroonium radicals.

. . R* and R are also especially a tyrosyl or lysyl radical.

In the case where Rj is an amine residue, symbolized by -NH-R, the residue R, which is therefore attached to the carboxyl by an amide bond, can be of any type but is preferably a residue suitable for labelling, for example with one or more radioactive atoms such as 12SI.

The chemical type of this residue R will pfbferablv inciude Λ x an amino acid, which will be used for bonding with the rest of the molecule of formula (I).

This amine residue will be for example a protein or a polypeptide containing from 2 to 10 amino acids and preferably 2 or 3 amino acids, or else a monoamino .3 such as tyrosine, or a diamino acid.

Said amino acids will preferably be natural amino acids or their amide derivatives; thus, for example, if the radical R^s a dipeptide consisting of a glycyl residue and a tyrosyl residue, it may be possible, for example, to replace the tyrosyl residue with a tyrosinamide residue.

The diamine may be used for the coupling of fluorophores or for the synthesis of dimers of formula (I'): R,R"N-A—B—0-CH2—CO-NH-R—NH-CO—CH2—Ο—B—A-N-R'R" (I* ) in which R, R* , R, A and B are as already defined.

Among the amine residues, there may be mentioned especially amino, hydrazino and nitrilo (-Ns) residues and the following peptides or peptide deriva25 tives: tyrosyl-cysteine, tyrosyl-cysteinamide, tyrosylglycine or tyrosyl-glycinamide.

There may also be mentioned protein derivatives joined to the carboxyl directly or via a peptide or an amino acid and via a heterobifunctional agent capable of reacting with a sulphydryl group and an amino group, such as SMCC, and the following residues may be mentioned as examples: tyrosyl-cysteinyl-protein, tyrosylcysteinyl-heterobifunctional agent such as (SMCC)protein, glycyl-tyrosine or glycyl-butane-1,4-diamine.

In the case of alcohol derived residues mention may be especially made of the phenol derivatives, optionally substituted on the phenyl ring, such as 2- or 4-nitrophenol, those derived from phenylmethanol which are unsubstituted or substituted on the phenyl by an alkyl radical, those derived from hydroxyalkyl trimethylsilyl, especially hydroxyethyl5 trimethylsilyl, and those derived from C4-Cw aliphatic alcohols.

Preferred derivatives according to the invention are derivatives of formula (I) as defined above which are characterized in that B is a phenyl nucleus, which is unsubstituted or substituted by one or more halogen atoms, or an indole nucleus, and their addition salts with mineral ' or organic acids. The nuclei can be substituted by a halogen atom, preferably chlorine, bromine or iodine, especially the indole nucleus, in which case substitution is preferably in the 2-position.

Among these derivatives in which B is an indole nucleus, it is particularly preferred to use those which are characterized in that R1 is a diamine, a protein, an amino acid or a polypeptide consisting of at most 5 amino acids, or derivatives of said amino acids or polypeptides, and especially tryptamine-5-Ocarboxymethylglycyltyros inamide [S-CM-GTNHa], as well as the derivatives mentioned in the Examples, and their addition salts with mineral or organic acids.

Among these derivatives, it is also preferred to use those which are characterized in that A is a radical -(CHa)a- and R' and R" are a linear or branched alkyl radical containing from 1 to 5 carbon atoms.

The present patent application further relates to a method of preparing the derivatives described above, characterized in that a derivative of formula (II): R'R’'N-Α-Β-ΟΗ (II) in which Rz, R", A and B are as already defined, is reacted with a derivative containing a protec5 ting group for the amine groups in the case where Rz = R - H, to give a derivative of formula < III): Y-HN-A-B-OH (III) in which A and B are as already defined and Y is a readily cleavable protecting group for the amine groups, which is reacted with a halogenoacetic acid to give a derivative of formula (IV): Y-HN—A—Β—O—CH2COOH (IV) in which A, B and Y are as already defined, which is reacted with an amine derivative or an alcohol ^ο give either the derivative· · of formula (I) as defined above, or if appropriate a dimer of formula (lz): R,RN-A-B-O-CH2-CO-NH-R-NH-CO-CH2-O-B-A-N-R#R (Iz ) in the case of a diamine, or a polymer in the case of a polyamine, which is isolated and, if desired, converted to a salt.

The derivatives of general formula I have a basic character. Salts of the derivatives of general formula I can advantageously be prepared, if appropriate, by reacting a mineral or organic acid with said derivative of general formula I in approximately stoichiometric proportions. If appropriate, the salts can be prepared without isolation of the corresponding bases.

The derivative capable of grafting a protecting group for the amine groups on to the derivative of formula (II) can be, for example, an acid chloride [9fluorophenylmethyloxycarbonyl chloride (FmocCl) or benzyloxycarbonyl chloride (BzCl)] or, preferably, an anhydride (di-t-butyl dicarbonate, (BOC)aO, or citra35 conic anhydride].

The derivative of formula (IV) is preferably prepared at alkaline pH in the presence of a metal oxide, such as magnesium oxide, and a halogenoacetic acid, it being possible for the latter to be a chlo5 ride, although it is preferably a bromide.

The reaction of the derivative of formula (IV) with the amine derivative (R^) is preferably carried out after activation of the carboxyl group in the form of a mixed anhydride by means of an alkyl chlorocarboxylate such as ethyl chloroformate. It is also possible to use acid chlorides and carbodiimides as activators or to form a hydrolyzable N-hydroxysuccinimide ester beforehand.

If necessary, the protecting group for the amine is cleaved by acid hydrolysis, especially with an - acid such as trifluoroacetic acid, or by alkaline hydrolysis, especially with piperidine.

The mediators of formula (II) and their synthetic analogues are well known from various publica20 tions and patents.

The derivative of formula (I), or one of its salts, can be coupled with a marker through the presence of a free carboxyl group in the residue R^ in formula I, as described for the bonding of the amine residue to the rest of the molecule of formula I.

If the marker is iodine, the ,xesidue_Kj will · contain a tyrosine (or a tyrosinamide) or a histamine.

If the marker is an enzyme, the residue R^· will contain carboxyl or sulphydryl groups.

If the marker is a fluorescent element, the residue R^ will contain a diamine.

Of the derivatives of formula I described above, we emphasize those which contain a protein grafted on to the O-carboxymethyl unit in accordance with the above methods.

The derivatives forming the subject of the present invention possess very valuable pharmacological properties. In particular, indole derivatives possess a remarkable affinity for serotonin receptors, especially the 5HT1D'S.

These properties are illustrated below in the experimental section. They justify the use of the mediator derivatives described above, and their addition salts with pharmaceutically acceptable acids, as drugs.

The drugs according to the present invention are employed for example in both the curative and preventive treatment of diseases associated with a dysfunction of the -HT receptors (particularly 5HT1D'S), their deregulation or modifications of the endogeneous ligand (generally serotonin).

They are employed in particular in the treat. ment of migraine.

The customary dose, which varies according to the subject treated and the complaint in question, can be for example from 0.1. to 10 mg per day, administered orally to man, of the derivative of Example 1.

The invention further relates to the pharmaceutical compositions which contain at least one of the afore-mentioned derivatives, or one of its addition salts with pharmaceutically acceptable acids, as the active principle.

As drugs, the derivatives of general formula I and their addition salts with pharmaceutically acceptable acids can be incorporated into pharmaceutical compositions intended for the digestive tract or for parenteral administration.

These pharmaceutical compositions can be solid or liquid, for example, and can be presented in the pharmaceutical forms commonly used in human medicine, such as, for example, simple or coated tablets, gelatin capsules, granules, sweets , suppositories, prepara10 tions for nasal instillation and injectable preparations; they are prepared in accordance with the customary methods. In said compositions, the active principle or principles can be incorporated into excipients normally employed in pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fats of animal or vegetable origin, paraffinic derivatives, glycols, various wetting agents, dispersants or emulsifiers, and preservatives.

As has been seen, the derivatives of formula (I) possess a substantial affinity for the receptors or else the carrier protein for the corresponding endogenous molecule.

The derivatives , forming the subject - of the * present patent application, which may or may not be labelled, for example with a radioactive element, especially "*I, or a fluorescent element or with an enzyme, are particularly useful for the visualization, both in vitro and in some cases in vivo, of endogenous mediator binding sites. These properties are illustrated below in the experimental sectiqn. A protein carrying a colloidal gold or a contrast medium can also be used as a marker.

The present patent application therefore further relates to the derivatives described above which are characterized in that they are in a labelled form.

The present patent application further relates to the use - . of the derivatives described above in the purification of endogenous mediator receptors.

The O-carboxymethylation of thyroid hormones modulates the interaction of these hormones with their carrier proteins. This property can be utilized to eliminate the binding of these native thyroid hormones to these car11 rier proteins. In the context of an immunoanalytical activity, the creation of analogues not recognized by the antibody, which inhibit binding to the carrier proteins, makes it possible to assay the total hormone.

The present patent application further relates to the application of the derivatives described above, and in particular the O-carboxymethylated derivatives of thyroxine, to the inhibition of binding to carrier proteins, for the purpose of assaying the total hor10 none.

The derivatives of the present patent application in which R-j ··. is a protein make it possible to prepare antibodies directed against the mediator corresponding to the rest of the molecule of formula (I).

The present patent application further relates - to the application of the derivatives described above to the visualization of endogenous mediator binding sites.

The present patent application further relates to the application of the derivatives of formula (I) to the preparation of antibodies directed against endogenous mediators..

The endogenous mediators [of formula (II), R'R-N-A-B-OH] or their derivatives may be assayed by means of these antibodies, using the derivatives of formula (I) as a tracer.

The present patent application finally relates to analytical kits, characterized in that they contain at least one of the derivatives of formula (I) des30 cribed above.

The Examples which follow illustrate the present invention without however implying a limitation.

EXPERIMENTAL SECTION Preparation I. Synthesis of thyroxine derivatives Series of examples of general formula I: R' R —N-A-B—O-CIL,—CO-R1 (I) in which R' = R = H, A = -CH(COOH)-CH2- and B is of the type -Bj-X-Ba-, where X = oxygen; if Bx - Ba = : derivatives of 3,3 * ,5,5' -tetraiodo-L-thyronine or L-thyroxine (T4); if Bx = and Ba = C^I: derivatives of 3,3z,5-triiodo-L-thyronine (T3).

Stage A 1. Synthesis of N-tert-butylcarbamate-O-carboxymethyl-T4 (BOC-T„-O-CHa-COOH) or -T, (BOC-Τ,-Ο-αςCOOH) 1.1. Protection of the amine group For the syntheses of Example 1, T4 or T3 is . used in the form of the free acid or the acid modified for example by amidation.

The amine group of T4 and T3 is protected with di-t-butyl dicarbonate [(BOC)2O] (Tarbell et al., Proc. 20 Nat. Acad. Sci. USA, 69, 1972, 730-732). 30 μΐ of triethylamine (TEA, 7.2 N) and 240 μΐ of 50 mM (BOC)aO in dimethyl sulphoxide (DMSO) are added to 10 μιηοΐ of T4 or T3. 1.2. Carboxymethylation of the hydroxyphenyl group The solution obtained above is mixed in equal volumes with a 500 mM aqueous solution of bromoacetic acid, with the pH adjusted to 12, in the presence of magnesium oxide and nitrogen gas. The mixture is stirred for 24 h in the dark. The medium is centrifuged for 10 minutes at 10,000 rpm. 1.3. Purification of the products by high performance liquid chromatography (HPLC) The supernatant is diluted in 5 volumes of 35 0.05% trifluoroacetic acid (TFA). One ml of this mix13 ture is injected into a column of μ Bondapack C14 grafted silica (10 pm, diameter 3.9 mm, length 30 cm). The derivatives of T3 are eluted isocratically for 30 min with a mixture of 50 vol. of 0.05% TFA and 50 vol. of methanol and then with a gradient reaching 100% of methanol in 60 minutes. For the derivatives of T4, the initial mixture for isocratic elution is composed of 40 vol. of 0.05% TFA and 60 vol. of methanol. The flow rate is 1 ml/min.

The starting materials (T3, T4) are collected during the isocratic period. The substituted derivatives are collected in the gradient: B0C-T3: 75% of methanol B0C-T3-O-CH2-C0OH: 78% of methanol BOC-T4: 80% of methanol BOC-T4-O-CH2-COOH: 82% of methanol (Figure 1) The fractions collected are analyzed by UV spectrophotometry. The absorption spectra in basic and acidic media are compared in order to determine whether substitution has indeed taken place on the hydroxyl group (Korman Clarke, op. cit.). The O-carboxymethylated derivatives do not show any spectral shift in basic media (Figure 1). The fractions containing the deriva25 tives BOC-T3-O-CHa-COOH and BOC-T4-O-CH2-COOH are evaporated and lyophilized. 2. Conjugation of the O-carboxymethylated derivatives with the radical R-NH2 2.1. Extension of the newly created side-chain An amide bond can be created between the carboxymethyl group and an amino acid, a peptide chain or a native or modified protein.

The carboxyl group of B0C-T3-0-CH2-CO0H or B0C-T4-0-CH2-C00H is activated by ethyl chloroformate (ECF, 7 pi, to which are added 7 pi of TEA in 5 ml of dimethylformamide). This solution is poured on to the lyophilizate in a volume such that the ECF is equimolar with the product to be activated. After activation for 5 min at 4*C, an equal volume of an aqueous solution of NH2-R, at a concentration 50 times greater than that of the carboxymethylated derivative, is added. NH2-R can be histamine, Gly-Tyr, Tyr-Gly, Cys or Gly-Cys, in the form of the free acid or the amide.

The products are separated by HPLC on a column of μ Bondapack Cle in a 0.05% TFA/methanol gradient. 2.2. Preparation of macromolecular derivatives 2.2.1. Grafting of the derivatives BOC-TS-OCH2-C0-NH-R on to proteins The derivatives as obtained in 1.3. or 2.1. above, or in the form of the free acid, are activated . by ethyl chloroformate (see 2.1.) and grafted on to a native protein (BSA, bovine serum albumin) or modified protein (Gly-BSA).

The derivatives BOC-T3-O-CHa-CO-NHR are separa20 ted by dialysis from the derivatives coupled with BSA, of the type BOC-Ts-O-CH2-CO-NH-R-BSA, where R - His, Gly-Tyr, Tyr-Gly, Cys, Gly-Cys.

The amine group of these derivatives can be deprotected in accordance with 3. 2.2.2. Grafting of the derivatives T3-0-CH2CONH-Cys or T3-0-CH2-C0NH-Gly-Cys on to proteins Proteins are modified by the addition of a heterobifunctional crosslinking agent of the N-hydroxysuccinimide type, for example succinimidyl-4-(N-male30 imidomethyl)cyclohexane-l-carboxylate (SMCC). The reaction (Ishikawa et al., 1983, J. Immunoassay, 4, 209-327) takes place at pH 7.3 in phosphate buffer. The protein whose NH2 residues are activated in this way is separated out by dialysis.

The derivatives T3-0-CH2-C0NH-Cys or T3-0-CH235 . CONH-Gly-Cys (amidated) are conjugated via the sulphydryl group to the modified proteins at pH 6.5.

The products are separated by dialysis to give derivatives of the general formula T3-O-CH2-CO-NH-Cys5 S—SMCC-BSA. 3. Preparation of O-carboxymethylthyroxine (T4-O-CH2-COOH) The amine group is deprotected by the addition of 200 μΐ of TFA (200 mg) to the derivative B0C-T3-010 CH2-C00H or BOC—T4—O—CH2—COOH, which has been lyophilized and cooled to -20*C. After a reaction time of one minute, the TFA is evaporated off under nitrogen gas.

Stage B 4. Iodination of the derivatives mCi of [^IJNa (2000 Ci/mmol, NEN) and 10 μΐ of chloramine T (CT, 1 mg/ml) are added to 1 nmol of Ts, BOC—T3 or BOC-Ts-O-CH2-COOH. After 90 sec, the reaction is stopped by 50 μΐ of sodium metabisulphite (SMB) over a period of 2 min. After the addition of 20 μΐ of methanol and stirring, the mixture is diluted in 1.5 ml of TFA (0.05%).

The reaction products are separated in accordance with the protocol described in A.l. for the deri25 vatives of Example 1. The products are excluded in the methanol gradient at the following respective concentrations: [125I]T4/ 62%; B0C[12sI]T4, 80%; BOCt^IJT,OCHjCOOH, 82%.

The radiolabelled derivatives sure diluted in methanol. The BOCt^IJ^-O-CHj-COOH is deprotected as indicated in 3.

EXAMPLE 1 . Synthesis of serotonin derivatives Series of examples of general formula I: R * R -N-A-B-O-C^-CO^ = R = H, A = —CH2—CH2— and Β = in which R* indole nucleus (OH in 5-position, aminoethyl in 3position). 1. Synthesis of N-tert-butylcarbamate-5-Οτ carboxymethyltryptamine [ BOC-S-CM ] Derivative of standard formula (IV): Y-HN-A-BO-CHa-COOH Stage A 1.1. Protection of the amine group The amine group of 5-HT is protected with di-tbutyl dicarbonate ((BOC)aO] (Tarbell et al., 1972). Equal volumes of 50 mM serotonin oxalate and 50 mM (BOC)aO in dimethyl sulphoxide (DMSO) are mixed in the presence of triethylamine (TEA). The reaction leading to BOC-S is instantaneous at roan temperature. 1.2. Carboxymethylation of the hydroxyphenyl group Protection with BOC is not necessary for certain serotonin derivatives which, like bufotenine, possess a tertiary amine, the residues R' and R in the general formula being methyl groups. As the rest of the molecule (residues A and B) is identical to serotonin, the O-carboxymethylation of bufotenine can be carried out directlyunder the same conditions as those described below for BOC-S.

The solution obtained in 1.1. above (Example 2) is mixed in equal volumes with a 500 mM aqueous solution of bromoacetic acid, with the pH adjusted to 12, in the presence of magnesium oxide and nitrogen gas.

The pH and the absence of oxygen and light are kept constant for 24 hours. The medium is centrifuged for 5 min at 10,000 rpm. 1.3. Purification by high performance liquid chromatography (HPLC) The derivative carboxymethyl-0-5-tryptamine-B0C (BOC-S-CM) is separated from the serotonin-BOC (BOC-S) and the oxidation products by HPLC.

The supernatant is diluted in two volumes of a 1% aqueous solution of trifluoroacetic acid. 1.8 ml of this mixture are injected in HPLC on to a column of Ultrasphere ODS Cie grafted silica (5 μπι, diameter 4.6 mm, length 15 cm) with isocratic elution (55 vol. of Η,Ο/0.05% TFA, 45 vol. of methanol). Elution is followed by spectrophotometry.

The carboxymethylation products of bufotenine 10 are separated on a column of μ Bondapack Cie, isocratic elution being carried out with 93 vol. of 0.05% TFA and vol. of acetonitrile.

The fractions collected are analyzed by spectrophotometry. The absorption spectra in basic and acidic media are compared in order to determine whether . substitution has indeed taken place on the hydroxyl group (Korman Clarke, op. cit.).

The fractions containing the derivative BOC-SCM are evaporated and lyophilized. 2. Conjugation of O-carboxymethylated derivatives with the radical R-NH2 2.1. Extension of the newly created side-chain An amide bond can be created with a peptide chain after activation of the acid group of BOC-S-CM by ethyl chloroformate (ECF).

The activating solution is composed of 5 ml of dimethylformamide (DMF), 7 μΐ of TEA and 7 μΐ of ECF. This solution is poured on to the lyophilized derivative BOC-S-CM in a volume such that the ECF is equi30 molar with the BOC-S-CM.

After activation for 5 minutes at 4*C, an equal volume of an aqueous solution of glycyl-tyrosinamide (or histamine, Tyr-Gly, Gly-Tyr, Gly-Gly or Gly-Cys, in the form of free or amidated acids), at a concentration so times greater than that of the BOC-S-CM, is added.

The reaction products are separated by HPLC on a column of Ultrasphere ODS Cie by isocratic elution (60 vol. of HaO pH 6, 40 vol. of methanol).

The fractions containing the derivative sero5 tonin-peptide (B0C-S-CM-GTNH2) are evaporated and lyophilized. 2.2. Preparation of macromolecular derivatives 2.2.1. Grafting of derivatives BOC-S-CM-R on to protein The derivatives as obtained in 1.3. or 2.1. above, or in the form of the free acid, are activated by ethyl chloroformate (see 2.1.) and grafted on to a native protein (BSA) or modified protein (Gly-BSA).

The derivatives BOC-S-CM-R are separated by dialysis from the derivatives coupled with BSA, of the . type BOC-S—CM—R—BSA.

The amine group of these derivatives can be deprotected in accordance with 3. above. 2.2.2. Grafting of derivatives S-CM-Gly-Cys on -0 to proteins Proteins are modified by the addition of heterobifunctional agents (see 2.2.2. of Example 1).

The derivative S-CM-Gly-Cys is grafted on to the above proteins via the sulphydryl group.

The products are separated by dialysis to give derivatives of the type S-CM-Gly-Cys-S-SMCC-BSA. 3. Deprotection of the amine group 200 μΐ of TFA (200 mg) are poured on to the derivative of the type BOC-S-CO-NHR (where R = H, Gly0 TyrNHa, His, Gly-TyrOH, Tyr-GlyNHa, Gly-GlyOH, GlyCys), which has been lyophilized and cooled to -20*C. After a reaction time of one minute, the TFA is evaporated off under nitrogen gas.

The reaction products are separated by HPLC on ’ a column of Ultrasphere ODS Cxe by isocratic elution (65 vol. of 0.05% TFA in water, 35 vol. of methanol).

The fractions containing the derivative possessing the free amine group (S-CM-GTNH,) are evaporated and lyophilized.

Stage B 4. Iodination of the derivatives S-CM-R If the radical R contains a histamine or a tyrosine, it is possible to carry out iodination with chloramine T. 1 mCi of 12SINa (2000 Ci/mmol, NEN) and 20 pi of CT (1 mg/ml) are added to 10 pi of S-CM-R (1 nmol in PBS medium). After 90 seconds, the reaction is stopped by 50 pi of 50 mM SMB. The mixture is diluted in 1.6 ml of 1% TFA.

The reaction products are separated by HPLC on a column of p Bondapack Cle by isocratic elution (72 'vol. of 0.05% TFA in water, 28 vol. of methanol).

The fractions obtained are counted by gamma spectrometry and diluted in a Krebs solution.

... I Characteristics of the binding of thyroxine derivatives to thyroxine binding proteins The derivatives of stage B of Preparation 1 (0.1 pi) are added to normal human serum (100' pi). Precipi25 tation is induced by 500 pi of dextran charcoal for 5 min at 4*C. After centrifugation for 10 min at 1000 rpm, the activity of the PELLET is counted.

The binding which persists relative to the initial binding is 99.9% for (iasI]T4, 80% for BOC[12SI3~ 30 T4-0-CH2-C00H and 75% for ["5I]T4-0-CH2-COOH.

II Study of the binding of serotonin derivatives to central receptors The test was performed with the product of Example 1, stage B.

The distribution of the receptor binding sites with a high affinity for serotonin (5-HTx) is hetero20 geneous in the central nervous system of the rat (Pazos and Palacios, Brain Research, 346, 1985, p. 205-230; S6gu et al.z Brain Research, 384, 1986, p. 205-217).

Furthermore, the content of subtypes of 5-HTx sites varies according to the anatomical structure in ques5 tion.

The protocol for preparing the brain sections is common to all the studies which follow. The animals are decapitated after anaesthesia with chloral hydrate (400 mg/kg of gross weight). The brains are quickly removed from the : skull - and frozen by immersion in isopentane cooled by liquid nitrogen. 20 pm sections, prepared in a cryostat at -20 'C, are mounted on gelatin-coated slides and stored at -20*C.

The sections are preincubated for 1 hour at 4*C in a Krebs solution (NaCl 118 mM; KC1 4.8 mM; CaCla 1.2 mM; MgCl2 1.2 mM; Tris-HCl 15 mM, pH 7.4) in order to remove the endogenous ligands. 1. Displacement, by the derivatives S-CM-R, of the high affinity binding of serotonin in rat brain sections 1.1. Protocol for incubation of the sections The incubations take place for 60 minutes at ’C in a Krebs solution containing 10 pM pargyline, .7.IO-4 M ascorbic acid, 2 nM [3H)5-HT (NEN, As = 30 Ci/mmol) and increasing concentrations of derivatives S-CM-R. The non-specific binding is determined on homothetic sections under the same conditions, but in the presence of 10-5 Μ 5-HT.

After incubation, the sections are rinsed three times for 20 seconds in distilled water and dried by a stream of hot air. The sections are affixed to a film (Amersham) for 6 weeks in the presence of a standard ([’H] microscale, Amersham). The films are developed for 6 minutes with Kodak D19, rinsed and fixed with AL4 (Kodak). Quantitative analysis of the autoradiographs is performed with an image analysis system (Sdgu et al., J. Neurosci. Methods, 31, 1990, p. 197-208). 1.2. Results The specific binding of [3H]5-HT is displaced in a one-phase process by 5-HT with an IC^ of 2 nM. S—O—CH2—COOH (S-CM) displaces it with an IC^ of 1000 nM and S-CM-Gly-TyrNHj and S-CM-Tyr-GlyNH, displace the binding in a two-phase process with IC^ values of 20 nM for the first site and 400 nM for the second. 2. Analysis of the binding sites for iodinated derivatives on rat central receptors 2.1. Protocol for incubation of the sections The incubations take place for 60 minutes at * C in a Krebs solution containing 10 μΚ pargyline, . 5.7.10-4 M ascorbic acid, 10 g/1 of bovine serum albumin (fraction V, Sigma) and 0.03 nmol/1 of the derivative S-CM-f^IJ-R. The non-specific binding is determined on homothetic sections under the same conditions, but in the presence of 10-« M 5-HT.

After incubation, the sections are rinsed twice for 1 min in distilled water and dried by a stream of hot air. The sections are affixed to a film (Amersham) for one week in the presence of standards (("«I] microscale, Amersham). The films are developed for 6 min with Kodak D19, rinsed and fixed with AL4 (Kodak). Quantitative analysis of the autoradiographs is performed with an image analysis system (S6gu et al., op. cit.). 2.2. Distribution of the labelling with S-CM[12SI]R fractions 2.2.1. Labelling with S-CM [ **«1] His The fractions of the preparation of the derivative S-CM [^1] His (A. 4.) do not label rat brain sections. 2.2.2. Labelling with S-CM-Gp^IJTNI^ Of the fractions collected during the preparation of the iodinated derivative of S-CM-GTNH, (A.4.), only the last fraction is retained specifically on rat brain sections. This fraction corresponds to S-CMG[*~I]TNH2.

Observation of the autoradiographs shows, in the mesencephalic region, intense labelling of the substantia nigra (SN) and the dorsal- subiculurij (SD) and no labelling of the hippocampus (H). This last structure is known for containing almost exclusively binding sites of the 5-HT^ type, whereas the others contain sites of the 5-HT1B type (Hoyer et al., Eur. J. Pharmacol., 118, 1985, p. 1—12). In the anterior region, the striatum (ST) is labelled: it contains especially 5„ HT^, sites? the choroid plexus (Px) , which contains -HT1<: sites, is )not labelled.

The conclusion which can be drawn is that S-CM-Gf^IJTNHj is a specific marker for binding sites with a high affinity for serotonin of the IB type. 3. Analysis of binding sites for S-CMGf^IJTNHj in the guinea-pig In the central nervous system of the guinea pig, the anatomical structures containing sites of the -HT^ type and the pharmacology of these sites are the same as in the rat. By contrast, the pharmacology of the sites contained in the substantia nigra of the guinea-pig is different from that of the sites of the same structure in the rat (Heuring and Peroutka, J.

Neurosci., 7, 1987, p. 894-903). These sites are therefore denoted as 5-HT1D.

With the same method of treating the sections as that used in 2. for the rat, it was demonstrated that in the guinea-pig (Figure 6), S-CM-Gt^IJTNIL, labels the substantia nigra (Figure 6C) and not the hippocampus (Figure 6B).

S-CM-G[l2SI]TNHa is a marker for sites with a high affinity for serotonin of the ID type.

This 5-HT derivative has a preferential affi5 nity for the 5-HT^ and 5-ΗΤιο sites compared with the -HT^ and 5-HTiC sites, making this ligand an important tool for analyzing receptors with a high affinity for 5-HT.

It is currently the only ligand to exhibit such 10 a high selectivity without binding to other receptors which are not of the 5-HT type (such as B-adrenergic receptors). Furthermore, it is the only ligand to label the 5-HT1D sites. It can therefore be used to study receptors of this type in man and their variation in neurodegenerative diseases (Huntington's chorea) . 4. Analysis of binding sites in the monkey The test was performed with the derivative of Example 2, stage A.

A. Biological preparation A male monkey (Macacca mulatta n.) weighing 8 kg was sacrificed by means of an overdose of barbiturates and exsanguination. Once the animal's death has been established, the . skull is removed and the brain is revealed. The right hemisphere is cut out and frozen by isopentane cooled in liquid nitrogen. The block is stored at -20 *C. 10 pm thick sections are prepared in a cryostat at -20 *C and placed on gelatincoated slides (Sdgu et al., 1990, J. Neurosci. Meth., 31, 197). They are stored at -20*C until required.

- Incubation with the radioactive probe The sections are preincubated for 1 h at 4*C in a Krebs solution (mM: NaCl, 118; KCl, 4.5; CaCl2, 1.2; MgCl2, 1.2; Tris, 15; pH 7.4) in order to remove the endogenous ligands. They are then incubated at 20*C for 60 minutes in a solution containing 105 M par24 gyline, 57 mM ascorbic acid, 10 g/1 of bovine serum albumin and 0.02 nM S-CM-Gt^IJTNHj, alone .. or in the presence of increasing concentrations of serotonin. Some of the sections are incubated under identical conditions except that the radioactive probe is replaced either with 2 nM pH]5-HT, or with 2 nM PH]5-HT in the presence of 100 nM 8-hydroxy-2-[di-Npropy 1 amino Jtetralin (8-OH-DPAT) and 100 nM mesulergin, or with 1 nM PHJ8-OH-DPAT. After rinsing 2-x^for 1 •10 minute in distilled water, the sections are dried.

- Autoradiography The slides are affixed to a tritium-sensitive film for 8 days. The film is developed for 6 minutes in D19, rinsed and fixed. The autoradiographs are quantified with a video system for image analysis (Sdgu et al., 1990).

B. Results Incubation in the presence of 2 nM pH] 5-HT (Figure 7A) shows substantial labelling of the hippo20 campus and the substantia nigra. Under these conditions, all the types of 5-HT^ sites are labelled.

Incubation in the presence of 1 nM pH]8-OHDPAT (Figure 7B), which only labels the 5-ΗΤ1λ sites, shows intense labelling of the hippocampus but not the substantia nigra.

In the case of incubation in pH]5-HT in the presence of 100 nM 8-OH-DPAT and mesulergin (Figure 7C), gc&x the 5-HT^ sites are labelled. The autoradiographs show a strong reaction with the substantia nigra.

Incubation in 0.02 nM S-CM-GC^I]TNHa by itself (Figure 7D) shows virtually exclusive labelling of the substantia nigra, as in the previous case. This Ocarboxymethylated derivative of serotonin binds to the -HT1D sites.

If the binding of 0.02 nM S-CM-GJ^IJTNI^ is displaced with serotonin, an ICM (concentration which inhibits 50% of the binding) of the order of 5 nM is obtained, therefore showing that the binding of the derivative is specific for the 5-HT^, sites.

Conclusion S-CM-G[125I]TNH2 is a marker for 5-HTxo receptors. Any analogous molecule will therefore have this property of binding to the 5-HTw sites.

. Analysis of the S-CM-G[12SI]TNHa binding sites in man a) Postmortem human brains are stored at -20*C. pm sections are· prepared from blocks containing either the locus niger or the Ammon's horn. The sections are treated as indicated in 4. for the monkey brain sections. b) After autoradiography, strong labelling is observed on the locus niger (structure corresponding to the substantia nigra in the rat) and weak labelling is observed on the Ammon's horn (equivalent in man to the hippocampus in the rat).

The locus niger contains virtually exclusively binding sites of the 5-HT1D type, whereas the Ammon's horn is composed predominantly of sites of the 5-HT^ type.

The iodinated derivative tested therefore labels the 5-HTw receptors of the human brain in vitro. Thus it can be used to study receptors of this type in man and their variation in neurodegenerative diseases (Huntington's chorea). The derivatives described in the present patent which have this property and which pass through the blood-brain barrier may be used for therapeutic purposes in the case of dysfunctions associated with the type of receptor in question (5-HTlo).

III Binding of serotonin derivatives to peripheral receptors 1. Passage across the blood-brain barrier If the molecules pass through the blood-brain barrier, they are capable of acting on the receptors of the central nervous system, an effect which has been researched in numerous cases (cf. point 4.).

In the case where receptors exist both in the peripheral nervous system and in the central nervous system, it may be desirable to have derivatives which do not pass through the blood-brain barrier. These derivatives will then have an effect on the peripheral receptors without a predominant central action.

A. Experimental protocol mice and 2 guinea-pigs are deeply anaesthetized with chloral hydrate (intraperitoneal injection of 0.12. ml of a 35% solution per 100 g of body weight). The rib cage is opened and the heart is revealed.

The mice and guinea-pigs receive intracardiac injections of 200 μΐ and 1.5 ml, respectively, of S-CMGC^IITNH,.

After 10 minutes, the animals, still under deep anaesthesia, are decapitated and the brains are frozen and stored as indicated in Example 1.

Sections are prepared and autoradiography is carried out as described in Example 1.

B. Results An examination of sections corresponding to different regions of the brain shows no labelling, particularly In the substantia nigra, an anatomical structure which is well labelled when incubation is carried out in vitro in the same tracer.

Conclusion The serotonin derivative tested does not cross- ‘the blood-brain barrier, making it possible to alleviate the most painful effects of migraine. No toxicity was observed at the dose injected. 2. study of the distribution of the binding of the derivative S-CM-G[12SI]TNH2 by visualization in vivo with a gamma camera Given that this derivative does not pass through the blood-brain barrier, as shown in 1., the . distribution of the peripheral labelling was studied by visualization in vivo.

The animals (mice) are anaesthetized and injected intravenously with the derivative S-CM-GC^IlTNHj. The product is very quickly observed to diffuse into the heart, the entire vascular system and the liver. minutes after the injection, the labelling decreases in the vascular system, except for the heart and the cerebral vascular system, in this latter zone, the labelling is retained for 15 to 20 minutes before slowly disappearing. The labelling of the bladder increases in the longer term, the heart remaining labelled for a fairly long time.

An experiment was performed with an iodinated derivative which exhibited the same specific binding as the above derivative but did not possess the property of binding specifically to the central 5-HT^ and 5-HTw sites. In this case, the decline in the labelling observed with the gamma camera is identical to that observed with S-CM-G[125I ]TNH2, except for the cerebral vascular system which does not exhibit any particular retention.

S-CM-GTNH2 and its derivatives can therefore be used for therapeutic purposes in the alleviation of migraine attacks. Being unable to pass through the blood-brain barrier, they are not capable of causing side-effects by acting on the central serotoninergic sites in roan.

EXAMPLE 3 Tablets having the following formulation were prepared: -O-carboxymethylglycyltyrosinamide (S-CM-GTNHJ .................................0.5 mg excipient q.s. for a finished tablet of......100 mg (details of the excipient: lactose, starch, talc, magnesium stearate) EXAMPLE 4 Divisible tablets having the following formulation were prepared: -O-carboxyraethylglycyltyrosinamide (S-CM-GTNHJ .................................. 2 mg excipient q.s. for a finished tablet of......100 mg (details of the excipient: lactose, starch, talc, magnesium ste.arate) EXAMPLE 5 An injectable preparation having the following formulation was prepared: 5-O-carboxymethylglycyltyrosinamide (S-CM-GTNH,) ................................. 2 mg excipient: water for injectable preparations . 2 ml EXAMPLE 6 A nasal aerosol having the following formulation was prepared: -O-carboxymethylglycyltyrosinamide (S-CM-GTNHJ ................................. 30 . mg excipient: aqueous solution: sodium chloride, trisodium citrate, citric acid distilled water.............................. 15 ml EXAMPLE 7 A buccal aerosol having the following formulation was prepared: -O-carboxymethylglycyltyrosinamide (S-CM-GTNHJ .................................. 60 mg excipient: aqueous solution: citric acid, ethyl alcohol, sweetener, flavouring, polysorbate 80, propylene glycol purified water............................... 50 ml propellant: nitrogen EXAMPLE 8: Kit for analyzing a 5-HTw receptor A kit was prepared for studying the presence of the 5-HT1D receptor and the affinity of derivatives for said receptor, and for revealing changes in the affi10 nity and number of these receptors, said kit having the following composition: - Product of Example 2, stage B (2000 Ci/mmol), in 50 mM Tris-HCl buffer pH 7.4 .................... 15 ml - Standard: lyophilized serotonin (oxalate), 10 nmol, and buffer pH 6.2 - Standard: product of Example 2, stage B, 1 nmol of serotonin per vial- , and buffer pH 6.2 - Inhibitor solution: 1.25 mmol of pargyline, 12.5 mmol of 8—OH-DPAT, 12.5 nmol of mesulergin per vial buffer pH 7.4 - Diluent: saline buffer pH 7.4 - Filters: glass fibre FIGURE CAPTIONS Figure 1: Purification of the carboxymethylation products of N-BOC-T, A: chromatogram after HPLC on μ Bondapack Cxe. 0-30 5 min: 50 vol. of 0.05% TFA, 50 vol. of methanol. 30-80 min: 100% methanol gradient.

The peak heights are not absolute because there are two different superimposed elution profiles.

B: absorption spectrum of BOC-T, in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from 220 to 350 nm.

The spectrum can be shifted in a basic medium, as for T3.

C: absorption spectrum of BOC-T3-OCH2-COOH in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from ,220 to 350 nm.

The spectrum cannot be shifted in a basic medium.

Figure 2: Purification of the carboxymethylation products of serotonin-N-BOC (BOC-S) A: chromatogram after HPLC on Ultrasphere ODS C„ with isocratic elution: 55 vol. of Ha0/0.05% TFA, 45 vol. of methanol. Ordinate: optical density (O.D.) for various wavelengths (pm) (indicated on the right). Abscissa : time in minutes.

B: absorption spectrum, adjusted at 280 nm, of the product emerging after 28 minutes, BOC-S (solid line), and the product emerging after 36 minutes, BOC-S-CM (dotted line). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

C: absorption spectrum of BOC-S-CM in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

D: absorption spectrum of serotonin in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D.

Abscissa: wavelengths from 200 to 350 nm.

E: absorption spectrum of BOC-S in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

Note that the spectrum is shifted in a basic medium, as 5 for serotonin (Figure ID). This is not the case with BOC-S-CM (Figure 1C), which is substituted on the OH. Figure 3: Purification of the conjugation products of BOC-S-CM and GTNH2 A: chromatogram after HPLC on Ultrasphere Cie with isocratic elution: 60 vol. of H2O pH 6, 40 vol. of methanol. Ordinate: O.D. for various wavelengths • (rim) (indicated on trie right). Abscissa : time in minutes.

B: absorption spectrum, adjusted at 280 nm, of the product emerging after 18 minutes, GTNH2 (solid line), the product emerging after 35 minutes, BOC-S-CM (dotted line), and the product emerging after 48 minutes, BOC. S-CM-GTNHa (dashed line). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

C: absorption spectrum of BOC-S in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

D: absorption spectrum of GTNH2 in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

E: absorption spectrum of BOC-S-CM-GTNHa in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D. Abscissa: wavelengths from 200 to 350 nm.

Note that the spectral shift in an alkaline medium does not correspond at all to that of a serotonin unsubsti30 tuted on the OH (Figure 2C), whereas the spectrum is shifted more than that of BOC-S-CM (Figure 1C). A shift towards the longer wavelengths is observed, as for GTNHa (Figure 2D), but this modification is smaller in terms of O.D.

Figure 4: Purification of the products resulting from deprotection of the amine of BOC-S-CM-GTNHa A: chromatogram after HPLC on Bondapack cie with isocratic elution: 65 vol. of HaO/0.05% TFA, 35 vol. of methanol. Ordinate: O.D. for 280 and 300 nm. Abs5 cissa: time in minutes.

B: absorption spectrum, adjusted at 280 nm, of the product emerging after 24 minutes, S-CM (solid line), and the product emerging after 30 minutes, S-CM-GTNH2 (dotted line).

The spectrum of the product emerging after 60 minutes, BOC-S-CM-GTNH,, is identical to that shown in Figure 2B.

C: absorption spectrum of S-CM—GTNHa in an acid medium (Ac) and an alkaline medium (Al). Ordinate: O.D.

Abscissa: wavelengths from 200 to 350 nm.

Note that the spectral shift as a function of pH is identical to that obtained for BOC-S-CM-GTNH,, shown in Figure 2E.

Figure 5: Analysis of the S-CM-G(12SI JTNH, binding sites in the rat - Rat brain sections (10 μτα) incubated in 0.03 nM S-CM-Gt^IJTNI^ A: «ulterior region Px: choroid plexus;-* ; St: striatum B: mesencephalic region SD: dorsal subic'uluni; SN: substantia nigra; H: hippocampus C: non-specific binding in the mesencephalic region, established in the presence of 10-5 Μ 5-HT Note that Px and H are not labelled (even though they respectively contain 5-HTlc and 5-HT^ sites) and that St, SD and SN (which possess 5-HT^ sites) are labelled.

Figure 6: Analysis of the S-CM-G[12SI]TNH2 binding sites in the guinea-pig - Guinea-pig brain sections (10 /xm) incubated in 0.03 nM S-CM-G[125I]TNH2 A: anterior region Px: choroid plexus ' ; st: striatum B: region of hippocampus SD: dorsal^s.ubiculun-; H: hippocampus C: region of substantia nigra SN: substantia nigra Note that Px and H are not labelled (even though they respectively contain 5-HTlc and 5-HT,^ sites) and that St, SD and SN (which possess 5-HTlB sites) are label10 led.

Figure 7: Detection of the 5-HTx receptors on monkey (Macacca mulatta n.) brain sections. The frozen tissue sections (10 pm) are incubated with various probes and subjected to autoradiography. h. Incubation in 2 nM [3H]5-HT, showing the total 5-HTx sites in the hippocampus (H) and the substantia nigra (SN).

B. Incubation in 1 nM [3H] 8-OH-DPAT, showing the 5-HT^ sites exclusively located in the hippocampus.

C. Incubation in 2 nM [3H]5-HT in the presence of 100 nM 8—OH-DPAT and 100 nM mesulergin, showing the 5-HT1B sites virtually exclusively located in the substantia nigra.

D. Incubation in 0.02 nM [12SI]S-CM-GTNH2, showing exclusive labelling of the substantia nigra, similar to that shown in C.

E. Same incubation as D, but with 10~s Μ 5-HT added, showing the non-specific binding (scale = 1 cm).

F. Diagram of the anatomical structures, taken from section All,5 of the atlas by Riche et al., 1968.

Claims (17)

1. The derivatives of biologically active molecules containing a 5 primary amine function and a hydroxylated nucleus as well as their addition salts with mineral or organic acids, characterized in that they correspond to general formula I: [R'RN-A-B-0-CH 2 -C0] n R 1 (I) in which n represents an integer from 1 to 10; A represents a linear or branched alkylene chain containing 1 to 5 carbon atoms; 15 B represents an aromatic nucleus containing 6 to 10 carbon atoms, if appropriate substituted and optionally containing a heteroatom; -Rj represents an amine residue, an alcohol residue chosen from the optionally substituted phenols and the Cj-Cjg aliphatic alcohols; and 20 R' and R represent an alkyl radical containing 1 to 5 carbon atoms or a hydrogen atom, as well as their addition salts with mineral or organic acids.

2. The derivatives of formula (I) as defined in Claim 1, as well as 25 their addition salts with mineral or organic acids, characterized in that B represents a phenyl or indole nucleus, optionally substituted by one or more halogen atoms.

3. The derivatives according to Claim 2 as well as their addition 30 salts with mineral or organic acids, characterized in that Rj represents an NHR radical in which R represents a diamine residue, a protein, an amino acid or a polypeptide constituted by at most 5 amino acids or derivatives of said amino acids or polypeptides. 35

4. The derivatives according to Claim 1, 2 or 3, characterized in that R' and R represent a linear or branched alkyl radical containing 1 to 5 carbon atoms, and A represents a -CHg-CHg radical, as well as their - 35 addition salts with mineral or organic acids.

5. One of the derivatives whose names follow: - tryptamine-5-0-carboxymethylglycyltyrosinamide [S-CM-GTNH 2 ], - tryptamine-5-0-carboxymethy1tyrosylglycinami de - as well as their addition salts with mineral or organic acids.

6. One of the derivatives whose names follow: - tryptamine-5-0-carboxymethylglycyliodotyrosinamide [S-CM-iodoGTN^] - tryptamine-5-0-carboxymethyliodotyrosylglycinamide - as well as their addition salts with mineral or organic acids.

7. The derivatives of biologically active molecules containing a primary amine function and a hydroxylated nucleus as well as their addition salts with mineral or organic acids, characterized in that they correspond to formula: R'RN-A-B-0-CH 2 -C0-NH-R-NH-C0-CH 2 -0-B-A-N-R'R in which A, B, R 1 and R have the meaning indicated in Claim 1, and R the meaning indicated in Claim 10, or their amides.

8. The derivatives according to any one of Claims 1 to 5, characterized in that they are presented in labelled form, the marker 125 can be a radioactive element, in particular I, a fluorescent molecule, an enzyme, a protein carrying a colloidal gold or a contrast medium.

9. The derivatives according to any one of Claims 1 to 5, characterized in that they contain a protein grafted onto the O-carboxymethyl member according to the process of Claim 10.

10. Preparation process for the derivatives of formula (I) as defined in Claim 1, as well as their addition salts with mineral or organic acids, characterized in that a derivative of formula (II): r'RN-A-B-OH (II) - 36 in which R', R, A and B have the meaning already indicated, is reacted with a derivative containing a protector group of the amine functions in the case where R' = R = H, in order to obtain a 5 derivative of formula (III): Y-HN-A-B-OH (III) in which A and B have the meaning already indicated and Y represents 10 an easily cleavable protector group of the amine functions, which is reacted with a halogenoacetic acid in order to obtain a derivative of formula (IV): Y-HN-A-B-0-CH 2 C00H (IV) _ in which A, B and Y have the meaning already indicated, which is reacted with an amine derivative or an alcohol derivative in order to obtain the derivative of formula (I) as defined above, if appropriate, in the form of a dimer of formula (I 1 ): R'RN-A-B-0-CH 2 -C0-R-NH-NH-C0-CH 2 -0-B-A-N-R'R (Γ) in the case of a diamine, or a polymer in the case of a polyamine, R representing the corresponding diamine or polyamine residue which is 25 isolated and, if desired, salified.

11. Pharmaceutical compositions characterized in that they contain at least one of the medicaments as defined in one of Claims 1 to 9 as active ingredient.

12. Use of the derivatives according to any one of Claims 4 and 5 for the visualization of endogenous mediator binding sites.

13. Use of the derivatives according to any one of Claims 5 to 8 for 35 the purification of endogenous mediator receptors.

14. Use of the derivatives according to Claim 9 for the preparation of antibodies directed against endogenous mediators and their derivatives. - 37

15. Analytical kits for determining mediators and their derivatives, characterized in that they contain at least one of the derivatives according to any one of Claims 1 to 9.

16. A derivative according to any one of Claims 1 to 9, substantially as described herein.

17. A preparation process according to Claim 10, substantially as 10 described herein.